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. 2016 Dec 1;311(6):F1253-F1259.
doi: 10.1152/ajprenal.00238.2016. Epub 2016 Sep 7.

Testosterone decreases urinary bladder smooth muscle excitability via novel signaling mechanism involving direct activation of the BK channels

Kiril L Hristov  1 , Shankar P Parajuli  1 , Aaron Provence  1 , Georgi V Petkov  2
Affiliations

Testosterone decreases urinary bladder smooth muscle excitability via novel signaling mechanism involving direct activation of the BK channels

Kiril L Hristov et al. Am J Physiol Renal Physiol. .

Abstract

In addition to improving sexual function, testosterone has been reported to have beneficial effects in ameliorating lower urinary tract symptoms by increasing bladder capacity and compliance, while decreasing bladder pressure. However, the cellular mechanisms by which testosterone regulates detrusor smooth muscle (DSM) excitability have not been elucidated. Here, we used amphotericin-B perforated whole cell patch-clamp and single channel recordings on inside-out excised membrane patches to investigate the regulatory role of testosterone in guinea pig DSM excitability. Testosterone (100 nM) significantly increased the depolarization-induced whole cell outward currents in DSM cells. The selective pharmacological inhibition of the large-conductance voltage- and Ca2+-activated K+ (BK) channels with paxilline (1 μM) completely abolished this stimulatory effect of testosterone, suggesting a mechanism involving BK channels. At a holding potential of -20 mV, DSM cells exhibited transient BK currents (TBKCs). Testosterone (100 nM) significantly increased TBKC activity in DSM cells. In current-clamp mode, testosterone (100 nM) significantly hyperpolarized the DSM cell resting membrane potential and increased spontaneous transient hyperpolarizations. Testosterone (100 nM) rapidly increased the single BK channel open probability in inside-out excised membrane patches from DSM cells, clearly suggesting a direct BK channel activation via a nongenomic mechanism. Live-cell Ca2+ imaging showed that testosterone (100 nM) caused a decrease in global intracellular Ca2+ concentration, consistent with testosterone-induced membrane hyperpolarization. In conclusion, the data provide compelling mechanistic evidence that under physiological conditions, testosterone at nanomolar concentrations directly activates BK channels in DSM cells, independent from genomic testosterone receptors, and thus regulates DSM excitability.

Keywords: lower urinary tract symptoms; overactive bladder; testosterone.

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Figures

Fig. 1.
Fig. 1.
Testosterone increases the depolarization-induced whole cell outward currents in freshly isolated detrusor smooth muscle (DSM) cells. A: representative original recordings illustrating the depolarization-induced whole cell outward currents in the absence (control) and in the presence of 100 nM testosterone. B: current-voltage relationship curve summarizes the stimulatory effects of 100 nM testosterone on the whole cell outward currents (n = 15, N = 6; *P < 0.05). C: representative original recordings illustrating the depolarization-induced whole cell outward currents in the presence of 1 μM paxilline alone and in the presence of both 1 μM paxilline and 100 nM testosterone. D: current-voltage relationship curve summarizes the lack of stimulatory effects of 100 nM testosterone on the whole cell outward currents in the presence of 1 μM paxilline (n = 7, N = 7; P > 0.05). The original recordings represented in A and C are from 2 separate DSM cells.
Fig. 2.
Fig. 2.
Testosterone increases transient BK currents (TBKCs) in DSM cells. A: representative original recording illustrating the stimulatory effect of 100 nM testosterone on the TBKC activity in an isolated DSM cell. A portion of the recording before (control) and after testosterone application is shown on an expanded time scale. B: summary data illustrating the stimulatory effects of 100 nM testosterone on TBKC frequency (n = 9, N = 9; *P < 0.05). TBKCs were recorded at a holding potential of −20 mV. The data were normalized to control values (before testosterone addition) taken as 100% and were presented in percentages (%).
Fig. 3.
Fig. 3.
Testosterone increases the single BK channel open probability (NPo) in inside-out excised membrane patches from DSM cells. A: representative original recording from an excised membrane patch of a DSM cell illustrating the stimulatory effect of 100 nM testosterone on NPo in inside-out configuration. Posttreatment of DSM cell membrane patches with 1 μM paxilline completely abolishes the single BK channel activity. “C” and “O” represent the closing and opening states of BK channels, respectively. B: summary data illustrating the stimulatory effects of 100 nM testosterone on NPo observed in inside-out excised patches (n = 13, N = 11; *P < 0.05).
Fig. 4.
Fig. 4.
Testosterone hyperpolarizes the resting membrane potential (RMP) and increases spontaneous transient hyperpolarizations (STHs) in DSM cells. A: representative trace of an RMP recording in current-clamp mode illustrating the hyperpolarizing effects of 100 nM testosterone in an isolated DSM cell. B: summary data illustrating the hyperpolarizing effects of testosterone on DSM cell RMP (n = 27, N = 21; *P < 0.05). C: summary data illustrating the effect of testosterone on STH amplitude and frequency (n = 9, N = 8; *P < 0.05). D: representative RMP recording in current-clamp mode demonstrating that when the BK channels are blocked with 1 μM paxilline, testosterone (100 nM) did not cause a membrane hyperpolarization in DSM cells. E: summary data illustrating that 100 nM testosterone had no effect on the DSM cell RMP in the presence of 1 μM paxilline (n = 6, N = 4; P > 0.05). NS, nonsignificant.
Fig. 5.
Fig. 5.
Testosterone reduces the intracellular Ca2+ levels in DSM cells. A: original recording showing a decrease in the global intracellular Ca2+ concentration by 100 nM testosterone in a DSM cell. B: summary data demonstrating the significant decrease in global intracellular Ca2+ concentration by 100 nM testosterone (n = 8, N = 5; *P < 0.05). Data are reported as the ratio of fura-2 AM fluorescent emission at 510 nm with excitation at 340 and 380 nm.
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